There is a critical need for new pharmaceutical agents against a wide variety of old and emerging human diseases, such as AIDS, antibiotic-resistant pathogens such as Mycobacterium tuberculosis, Staphylococcus aureus, and Pseudomonas aeruginosa, fungal diseases, and many types of cancer. However, the rate at which new types of compounds are being discovered has slowed. Natural products (chemicals made by living organisms such as bacteria and fungi) have historically been the source of most of our medicines. The natural world provides multiple examples of novel chemical classes with medically relevant biological activities. One is exemplified by the cyclic peptide toxins of fungi in the genus Amanita. These compounds have several unique and desirable properties among known natural products that make them promising scaffolds on which to design new therapeutic compounds for improving human health. For example, they are the smallest known peptides synthesized on ribosomes, and some of them already are known to have strong and unique biological targets. Methods used in the proposed studies include molecular genetics, next-generation deep genome sequencing, bioinformatics, biochemistry, chemistry, and bioassays against target organisms and mammalian cancer lines. The specific aims of this proposal are, first, to identify all of the steps in the biosynthesis of the amatoxins and phallotoxins, which are predicted to include proteolytic processing, cyclization, hydroxylation, synthesis of a unique amino acid crossbridge (trypathionine), and, in the case of the phallotoxins, conversion of one amino acid from the L to the unusual D form. The second aim is to study the biosynthesis of the amatoxins in Galerina, a mushroom unrelated to Amanita that makes the same compounds. The work on Amanita and Galerina will complement each other in several ways, leading to a more definitive description of the biosynthetic pathway. The third aim is to express the toxin biosynthetic pathway in another host organism that is more genetically tractable than Amanita itself. This will establish an experimental platform to dissect the pathway in detail, and to modify it in beneficial ways. The fourth aim is to use this experimental system to synthesize in vivo new cyclic peptides based on the Amanita toxin scaffold. There are more than 3 million possible permutations of amanitin, and this library of novel compounds is predicted to be rich in chemicals of potential pharmaceutical importance for treatment of bacterial and fungal diseases, and cancer.